The Diagnostic Value of LINC00426 in Type 2 Diabetes and Diabetic Kidney Disease and its Regulatory Effects on Renal Cells

Summary

Downregulation of LINC00426 promotes the progression of diabetic kidney disease (DKD) by inducing apoptosis in renal cells and increasing inflammation.

Abstract

The study investigates the diagnostic value of LINC00426 in type 2 diabetes (T2DM) and diabetic kidney disease (DKD), along with its regulatory effects on renal cells. The study included 280 T2DM patients (146 cases of DKD), and 186 healthy controls who visited during the same period. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) confirmed that serum LINC00426 is downregulated in patients with T2DM and DKD. Receiver operating characteristic (ROC) analysis assessed LINC00426’s diagnostic value for T2DM and DKD. Multivariate logistic regression identified independent DKD risk factors. LINC00426 showed diagnostic value for both diseases and negatively correlated with the urine albumin-to-creatinine ratio (UACR). Its downregulation independently predicted DKD development. A high-glucose (HG) environment inhibits LINC00426 expression in HK-2 and HGMC cells. CCK8 assay, flow cytometry, and enzyme-linked immunosorbent assays confirmed that HG also suppressed proliferation while promoting apoptosis and inflammation. In contrast, overexpression of LINC00426 enhances the proliferative capacity of HK-2 and HGMC cells, inhibits apoptosis, and reduces inflammatory responses. Downregulation of LINC00426 may exacerbate DKD progression by inhibiting the proliferation of renal cells (HK-2 and HGMC) and promoting apoptosis and inflammatory responses.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder¹. Reports indicate that T2DM and its complications have become a significant global public health concern^2,3, posing threats to human health and societal development. Due to unhealthy lifestyle habits acquired over time⁴, the prevalence of T2DM has been increasing annually⁵. Diabetic nephropathy (DN) was renamed diabetic kidney disease (DKD) in 2007^6,7. As a common complication of T2DM⁸, its incidence and mortality rates among diabetic patients continue to rise annually⁹. DKD often remains latent in patients with long-standing diabetes¹⁰ or manifests abruptly¹¹. DKD can cause kidney dysfunction and endothelial damage^12,13. DKD primarily manifests as the urinary loss of large molecules such as proteins following renal filtration system damage ¹⁴. However, for patients, blood biochemical markers (fasting glucose, insulin) serve as the gold standard for diagnosing T2DM, yet DKD remains difficult to detect. Conventional treatment approaches fail to promptly identify or assess DKD progression¹⁵. Therefore, raising awareness of the disease and identifying novel diagnostic biomarkers is crucial for the prevention and treatment of both T2DM and DKD.

In recent years, the involvement of long non-coding RNA (lncRNA) in diverse functions such as immunity¹⁶, cancer regulation, and metabolism has become increasingly recognized¹⁷. Accumulating evidence now implicates dysregulated lncRNA expression in the pathogenesis of complex chronic diseases, notably DKD^18,19. For instance, Zhang et al.²⁰ identified through high-throughput sequencing that lncRNA evf-2 interacts with hnRNP, promoting DKD development by enhancing expression of cell cycle-related genes and inflammatory factors. In a DKD rat model, lncRNA USR0000B2476D was identified as involved in immune regulation²¹. LINC00426 is a newly discovered lncRNA that plays an important role in the regulation of inflammatory factors²². Recent comparative microarray studies suggest LINC00426 may hold potential as a biomarker for T2DM development²³. Furthermore, bioinformatics strategies have captured LINC00426's involvement in immune regulation within renal cell carcinoma²⁴. Numerous studies have also highlighted the pivotal involvement of inflammation in diabetes and DKD²⁵ in diabetes and DKD. Existing studies have shown that T2DM²⁶ and prediabetes-related conditions²⁷, including metabolic syndrome²⁸, obesity²⁹, and cardiovascular metabolic heart disease³⁰, are all associated with a high inflammatory burden. In addition, diabetic microvascular complications, such as neuropathy³¹, nephropathy³², and retinopathy³³, as well as macrovascular complications³⁴, are all chronic inflammatory responses. Therefore, studying the relationship between LINC00426 and T2DM and DKD is very important for discovering new diagnostic markers and revealing the mechanisms of DKD. Nevertheless, current literature regarding the role of LINC00426 in TD2M and DKD remains limited, with no clinical evidence clarifying its expression pattern, independent risk value, and diagnostic efficacy in DKD patients.

To fill this research gap, the present study enrolled 466 participants, detected serum LINC00426 expression via RT-qPCR, identified independent risk factors for DKD using logistic regression, and further evaluated the diagnostic potential of LINC00426 for T2DM and DKD by ROC analysis. Our findings preliminarily clarify the expression characteristics and clinical significance of LINC00426 in DKD, thereby filling the existing knowledge gap and providing new insights into DKD pathogenesis and biomarker exploration.

Protocol

This study has been approved by the Medical Ethics Committee of The Third Xiangya Hospital of Central South University. All patients and their families expressed support and signed written informed consent forms.

Sample Collection
The sample size was calculated using G*Power software. The significance level (α) was set at 0.05, statistical power (1-β) at 0.8, effect size (f) at 0.25, and a 10% dropout rate was considered. The calculation results indicated that at least 123 participants per group were required when studying the control group, T2DM, and DKD populations. In the actual study, 186 participants were included in the control group, 134 patients in the T2DM group, and 146 patients in the DKD group. All were volunteers who received treatment at The Third Xiangya Hospital of Central South University from July 2022 to June 2023. Among these, 72 patients had no complications, whereas 208 patients presented with one or more complications. The complications primarily included diabetic neuropathy, diabetic retinopathy, and diabetic kidney disease (DKD), with 146 patients diagnosed with DKD. Diabetes classification was determined based on fasting insulin and fasting blood glucose levels; patients with type 1 diabetes were excluded to avoid diagnostic ambiguity. Cases of gestational diabetes and special diabetes conditions were excluded.

The inclusion criteria were as follows: Age ≥ 18 years; complete medical records and documentation available; compliance with the 2020 Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes³⁷. Exclusion criteria included comorbid psychiatric disorders, cognitive impairment, or inability to cooperate with the study; acute infectious conditions; organ failure; and malignant tumors.

A control group of 186 healthy individuals undergoing routine medical examinations during the same period was selected. Control group participants must meet the following criteria: no history of diabetes or other metabolic disorders; normal fasting blood glucose (<6.1 mmol/L), 2-hour postprandial glucose (<7.8 mmol/L), and glycated hemoglobin (<5.7%); and no history of renal disease or complications, with normal renal function indicators (glomerular filtration rate, urine albumin/creatinine ratio).

Diabetes-Related Indicator Testing
Basic demographic information was recorded for each study participant upon admission. The following morning, 5 mL of venous blood was collected from each subject in a fasting state for testing biochemical indicators. Fasting blood glucose (FBG) levels were analyzed using a blood glucose meter³⁵. Glycated hemoglobin (HbA1c) was measured with a fully automated HbA1c analyzer³⁶. Urine samples were collected within 24 hours of study inclusion. Urinary albumin concentration was measured using a protein analyzer³⁷ via the immune-turbidimetric method³⁸. The degree of renal impairment in DKD patients was assessed by the urinary albumin-to-creatinine ratio (UACR), a well-established index for assessing early renal injury and disease progression in DKD^3,39.

Cell Culture and Processing
Human renal epithelial cells HK-2 and glomerular mesangial cells HGMC were purchased. Specifically, HK-2 cells were cultured in K-SFM medium containing 10% fetal bovine serum, supplemented with 1% antibiotic solution. HGMC cells were cultured in primary cell complete medium. Cells were maintained in a 37 °C, 5% CO₂ incubator. Passaging and transfection were performed when confluence exceeded 70%. For high-glucose (HG) induction, cells were seeded in 6-well plates and exposed to 30 mM D-glucose in 2 mL of culture medium per well for 48 h (HK-2⁴⁰ and HGMC cells⁴¹). A low-glucose control group was treated with 5.5 mM D-glucose + 24.5 mM mannitol.

The full-length sequence of LINC00426 was cloned into the eukaryotic expression plasmid pcDNA3.1 to construct the pcDNA3.1 LINC00426 overexpression plasmid. The empty pcDNA3.1 vector was used as the negative control. Cell transfection was performed using Lipofectamine 3000 reagent according to the manufacturer’s instructions. Briefly, HG-induced HK-2 and HGMC cells were seeded into 6-well plates and transfected at 70%–80% confluence. For each well, 2.5 µg of plasmid DNA (pcDNA 3.1 or pcDNA3.1 LINC00426) was mixed with 5 µL of Lipofectamine 3000 reagent in 250 µL Opti-MEM for 15 min at room temperature according to previous research¹⁰. The transfection complex was then added dropwise to cells in complete culture medium. After 6 h of incubation at 37 °C, the medium was replaced with fresh medium, and cells were cultured for an additional 42 h before subsequent experiments.

Total RNA Extraction and Reverse Transcription
The remaining blood was centrifuged again at 3000 x g for 15 min, and the supernatant was collected and stored at -80 °C. Total RNA was extracted from serum and cell samples using the Trizol-phenol-chloroform method, precipitated with equal-volume ethanol according to previous research⁴². RNA concentration and purity were assessed using a UV spectrophotometer. Total cDNA was synthesized from samples using the RT reagent mix via a gene PCR amplifier. Reaction conditions were 42 °C for 15 min, followed by 85 °C for 5 s. Samples were stored briefly at 4 °C.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)
RT-qPCR was performed using SYBR qPCR master mix. Serum LINC00426 expression was quantified via the quantitative PCR instrument. Data was normalized using β-tubulin as the internal reference gene, calculated via the 2^−ΔΔCt method. The average Ct value of β-tubulin was approximately 25, ranging from 23 to 27 across all serum samples. The Ct coefficient of variation (CV) was 2.5%. One-way ANOVA showed no significant difference in β-tubulin Ct values among the control, T2DM, and DKD groups, confirming its stable expression and suitability as an internal reference for serum LINC00426 normalization. The primers involved in the reaction are listed in Supplementary Table 1. Reaction conditions: pre-denaturation at 95 °C for 10 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; final extension at 60 °C for 15 s.

Western blot analysis
Western blot analysis was performed following our established experimental protocols. Briefly, cells were lysed with RIPA lysis buffer supplemented with 1% protease inhibitor cocktail. After incubation at room temperature for 30 min, protein quantification was conducted. Protein samples were denatured at 95 °C, and equal amounts of 30 µg protein were separated by SDS-PAGE, followed by electrotransfer onto 0.45 µm PVDF membranes. Membranes were blocked with non-fat milk at room temperature for 2 h. Primary antibodies against Bax, Bcl-2, Caspase-3, IκBα, and p-P65 were diluted at 1:1000 with TBST, while β-actin was diluted at 1:3000. Membranes were incubated with primary antibodies overnight at 4 °C. Subsequently, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using an ECL substrate on a gel imaging system. Band gray values were quantified with ImageJ software.

Cell Counting Kit-8 (CCK8)
Digest transfected cells with 0.25% trypsin (EDTA-free) for approximately 3 min, then centrifuge at 800 x g for 5 min. Collect the pellet and resuspend in cell culture medium. Seed 100 µL of cell suspension (2 x 10³ cells) into a 96-well plate. After incubation for 24 h at 37 °C with 5% CO₂ to allow cell adherence, add 10 µL of CCK8 solution. The initial addition of CCK8 solution is designated as 0 h. The absorbance at 450 nm is measured using a microplate reader at 0, 24, 48, and 72 h.

Flow Cytometry
The effect of transfection on cell apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining solution. After transfection, cells were digested using the same trypsin digestion procedure as described above. The cells were washed with phosphate-buffered saline (PBS) and resuspended. Cell pellets were collected by centrifugation at 800 x g for 5 min. Transfer 100 µL (approximately 1 x 10⁵ cells) to a flow cytometry tube, centrifuge at 800 x g for 5 min at room temperature and discard the supernatant. Resuspend cells in 195 µL Annexin V-FITC binding buffer. Add 5 µL Annexin V-FITC and 10 µL PI staining solution to the mixture, then mix thoroughly. Incubate in the dark at room temperature for 20 min. Subsequently, place the treated flow cytometry tube in an ice bath for temporary storage. Before analysis, filter the cell suspension through a 200-micron mesh filter and resuspend. Collect cell signals using the flow cytometry system under excitation at 488 nm/636 nm.

Enzyme-linked immunosorbent assay (ELISA)
Cell inflammatory cytokines were measured using commercially available ELISA kits for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) IL-1 beta (IL-1β). The specific detection procedure is as follows: Add 100 µL of the appropriately diluted standard to the standard wells. Add 100 µL of cell basal medium to all blank wells. Add 50 µL of cell supernatant and 50 µL of 1 x detection buffer to the sample wells. Subsequently, add 50 µL of the detection antibody solution to the appropriate wells. Cover the microplate and incubate at room temperature for 2 h. Discard the liquid from the wells and wash 6 times with wash buffer. To achieve optimal results, ensure the wells are thoroughly dried. Add 100 µL of streptavidin working solution to each well again, seal the plate, and incubate at room temperature for 45 min. Finally, add 100 µL of chromogenic substrate, then incubate them in the dark for 20 min. Then add 100 µL of stop solution and gently mix. The solution will now appear as a clear, yellowish liquid of varying intensity. Measure the optical density (OD) of the solution at 450 nm and 630 nm using a microplate reader. The difference between these two values corresponds to the concentration of the inflammatory factor in the sample.

Statistical Analysis
Data was processed and analyzed using commercially available statistical software. All experimental results comprised three valid replicates. Intergroup comparisons employed independent samples t-tests and analysis of variance (ANOVA). Multiple comparisons were performed using the Tukey method. The ROC curve was used to evaluate the diagnostic value of LINC00426 for T2DM and DKD. Logistic regression analysis assessed risk factors for DKD. P < 0.05 indicated statistical significance for the respective data results.

Results

Comparison of Baseline Characteristics Between Healthy Individuals and T2DM Patients
The study enrolled 280 T2DM patients and 186 healthy individuals as controls. Analysis of the participants' medical records (see Table 1) revealed that the mean age of the control group was 58.91 ± 17.63 years, compared to 59.50 ± 17.14 years for the T2DM patients. However, there were no significant differences in gender or obesity between the two groups of patients. Further comparison revealed that,...

Discussion

DKD represents a severe microvascular complication in T2DM. Its earliest manifestation is the presence of microalbuminuria; detecting this in the early stages could potentially delay DKD progression. However, microalbuminuria lacks sensitivity in routine urine analysis and is consequently frequently overlooked⁴³. This study found that LINC00426 is downregulated in T2DM patients and possesses diagnostic value for both T2DM and DKD. Furthermore, LINC00426 may prevent DKD by reducing apoptosis in HG ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin	Gibco	25200056
Annexin V-FITC Apoptosis Detection Kit	Beyotime	C1062S
Antibiotics for cell culture	Gibco	R-015-10
Aristo specific protein detector	GOLDSITE	GPP-100
Bax Antibody	Cell signaling Technology	2772
BCA protein assay kit	Beyotime	P0013B
Bcl-2 (124) Mouse Monoclonal Antibody	Cell signaling Technology	15071
beta-Actin (13E5) Rabbit Monoclonal Antibody	Cell signaling Technology	4970
Blood glucose monitor	Yuwell	710
Caspase-3 Antibody	Cell signaling Technology	9662
CCK-8 reagent	Sigma	96992
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711
Chloroform	Sigma-Aldrich	C2432
Clarity Western ECL Substrate	Bio-Rad	1705061
Constant-temperature cell incubator	SaiLun	DHP-9032
D-Glucose	MedChemExpress	HY-B0389
Flow cytometer	Beckman coulted	CytoFLEX	Chloroform
Fluorescent quantitative PCR instrument	Roche	Light Cycler 96
Foetal bovine serum	Sigma-Aldrich	F0193
Fully automated glycated haemoglobin analyser	Hanyu medical	HLC-723G11	Phenol
Gene PCR amplifier	Bio Rad	S1000
Glomerular mesangial cells, HGMC	Sunncell Biotechnology Co., Ltd.	SNP-H252
Human Basic IL-1 beta ELISA Kit	Invitrogen	ECH002
Human IL-6 ELISA Kit	Invitrogen	EH2IL6
Human kidney epithelial cells, HK-2	Sunncell Biotechnology Co., Ltd.	SNL-165
Human TNF alpha ELISA Kit	Invitrogen	KAC1751
IkappaB alpha (L35A5) Mouse Monoclonal Antibody	Cell signaling Technology	4814
K-SFM medium	Invitrogen	17005-042
Lipofectamine 3000	Invitrogen	L3000015
Mannitol	Sigma-Aldrich	M9647
Multifunctional enzyme-linked immunosorbent assay reader	TECAN	GENios Plus
NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody	Cell signaling Technology	8242
non-fat milk	Biosharp	BS102-500g
Opti-MEM™ I Reduced Serum Medium	Invitrogen	31985062
P3000 Reagent	Invitrogen	L3000-015
PBS buffer	Sangon Biotech	E607080
Phenol	Sigma-Aldrich	P1037
Phospho-NF-kappaB p65 (Ser536) (93H1) Rabbit Monoclonal Antibody	Cell signaling Technology	3033
Primary cell complete medium	Sunncell Biotechnology Co., Ltd.	SNPM-H252
Prime Script RT reagent Kit	Takara	RR037A
PVDF membranes	Merck Millipore	SE1M003M00
rotease and phosphatase inhibitor cocktail	Beyotime	P1045
Trizol	Invitrogen	15596026CN
Ultraviolet spectrophotometer	Thermo	Nanodrop 2000